Method and kit for detecting FecB site polymorphism of sheep BMPR1B gene based on molecular beacon-melting curve and application of method and kit

Abstract

The invention provides a method and a kit for detecting FecB site polymorphism of a sheep BMPR1B gene based on a molecular beacon-melting curve and application of the method and the kit. And the FecB site polymorphism is generated by A > G mutation at 746nt of a BMPR1B gene coding region. The method comprises the following steps: preparing a fluorescent quantitative PCR reaction system by adopting a specific primer and molecular beacon probe composition, and judging that the genotype is C.746nt AA type when a melting curve peak is 53-56 DEG C; when the peak of the melting curve is 46-49 DEG C, judging that the genotype is C.746nt GG type; and when the two melting curve peaks exist, judging that the genotype is a C.746nt AG type. According to the method disclosed by the invention, whole-course tube closing operation is realized, nucleic acid aerosol pollution caused by uncovering sample application in a gel electrophoresis test is avoided, and the accuracy of a detection result is ensured. The real-time fluorescence PCR instrument is used for monitoring the fluorescence change in the amplification process and the fusion curve process in the whole process, the genotype result judgment of the FecB site can be completed after the test is finished, and the method is simple, convenient and rapid and is suitable for being applied to large-scale detection.

Description

Technical Field

[0001] The present invention relates to the fields of sheep reproduction identification and breeding, and particularly relates to a method, kit and application for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene based on molecular beacon-melting curve. Technical Background

[0002] The sheep BMPR1B gene is one of the known major genes for high fecundity in sheep. The FceB locus of this gene has been confirmed to be significantly associated with the reproductive traits of multiple sheep breeds. The FecB locus is generated by an A>G mutation at 746nt in the coding region of this gene, which can cause the amino acid at position 249 of the encoded protein to change from glutamine to arginine. It is a causal mutation affecting the multiple-ovulation trait in sheep. Among them, Fec B is the major gene for the multiple-ovulation trait. Fec B That is, G at 746nt of the BMPR-IB gene, Fec + That is, A at 746nt of the BMPR-IB gene. Two Fec B carriers are denoted by Fec B Fec B and abbreviated as BB; carriers with one Fec B copy are denoted by Fec B Fec + and abbreviated as B+, and non-carriers are denoted by Fec + Fec + and abbreviated as ++. One Fec B copy can increase the number of ovulations by 1.3 - 1.6, and two Fec B copies can increase the number of ovulations by 2.7 - 3.0. When reflected in production, the litter size of ewes carrying one Fec B copy increases by 0.9 - 1.2, and the litter size of ewes carrying two Fec B copies increases by 1.1 - 1.7. At present, the FecB locus has been successfully used in the breeding of new multiple-ovulation varieties (lines) of multiple sheep breeds and has become an effective genetic marker for marker-assisted selection (MAS).

[0003] Currently, there are mainly three categories of methods for detecting the FecB locus. One is the amplification method based on specific primers: different primers are designed according to the different nucleotides at the mutation site, and after amplification, the bands are observed by gel electrophoresis to judge; the second is to select a restriction endonuclease to digest the amplified product according to the sequence characteristics of the FecB locus by searching for restriction sites, and then the bands are observed by gel electrophoresis to judge; the third is to directly amplify the gene fragment containing the FecB locus and use Sanger sequencing to judge according to the sequencing peak map.

[0004] In current common detection methods, most are based on gel electrophoresis tests after primer amplification for judgment, or directly display the nucleotides at this site with the help of first-generation sequencing technology. Such as the detection technology shown in Chinese Patent Application CN 117187401 A.

[0005] Considering the technological generation gap: The existing detection and determination based on first-generation PCR requires visual observation through gel electrophoresis, with poor sensitivity; in addition, only two upstream and downstream primers are used to complete the amplification and determination, and compared with multiple primer or primer + probe amplification combinations, the specificity is poor. Considering operation accuracy and convenience: The existing method is undoubtedly more complex in operation after PCR and then through gel electrophoresis tests for detection and determination. At the same time, multi-step operations increase the uncertainty of test results. And Sanger sequencing of the amplicons after amplifying the target fragment significantly increases the test cost and cycle, and is extremely unsuitable for large-scale clinical or first-line rapid detection. Summary of the Invention

[0006] In view of this, in order to overcome the deficiencies of the prior art, the present invention provides a specific primer and molecular beacon probe composition for detecting the polymorphism of the FecB site of the sheep BMPR1B gene. The composition includes an upstream primer, the nucleotides of which are shown in SEQ ID NO.1; a downstream primer, the nucleotides of which are shown in SEQ ID NO.2; a molecular beacon probe, the nucleotides of which are shown in SEQ ID NO.3; the 5' end of the molecular beacon probe is labeled with a fluorescent group and the 3' end is labeled with a quenching group; the polymorphism of the FecB site is generated by the A>G mutation at 746nt in the coding region of the BMPR1B gene.

[0007] Further, the 5' end of the molecular beacon probe is labeled with a FAM fluorescent group and the 3' end is labeled with a BHQ1 quenching group.

[0008] The present invention also provides a method for detecting the polymorphism of the FecB site of the sheep BMPR1B gene based on molecular beacon-melting curve, and the method includes the steps:

[0009] 1) Extract the total DNA of the detection sample;

[0010] 2) Prepare a fluorescence quantitative PCR reaction system with any of the above compositions, and add the total DNA of the sample for detection;

[0011] 3) Set the reaction conditions;

[0012] 4) Analyze the melting curve after the reaction;

[0013] 5) Judge the genotype of the FecB site of the sheep BMPR1B gene according to the melting curve peak.

[0014] Further, the reaction conditions are as follows: pre-denaturation at 95°C for 180 s, 1 cycle; PCR at 95°C for 10 s, 60°C for 30 s, 72°C for 30 s, 40 cycles; denaturation at 95°C for 120 s, 1 cycle; annealing at 40°C for 120 s, 1 cycle; melting curve at 40°C - 65°C, 1 cycle.

[0015] Further, when the melting curve peak is at 53 - 56°C, the genotype is determined to be C.746nt AA type, i.e., the ++ locus; when the melting curve peak is at 46 - 49°C, the genotype is determined to be C.746nt GG type, i.e., the BB locus; when there are the above two melting curve peaks, the genotype is determined to be C.746nt AG type, i.e., the B+ locus.

[0016] The detection sample is sheep ear tissue or whole sheep blood.

[0017] The present invention also provides a kit for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene based on the molecular beacon - melting curve method, and the kit contains the above composition.

[0018] Further, the kit also includes an enzyme mixture and a positive sample control; the enzyme mixture contains: Taq enzyme, dNTP and Mg 2+ .

[0019] The present invention also provides the application of the method for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene in the screening of genetic markers for high fecundity in sheep.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. By designing a molecular beacon probe, the present invention makes it applicable to the high-resolution melting curve technology. Then, by designing a pair of primers to amplify the sequence covering the FecB locus, the fluorescence signal can be collected in a short time by utilizing the characteristic of the molecular beacon probe specifically binding to the amplified target sequence. Since different Tm values are generated when the molecular beacon probe has a completely complementary sequence and an incompletely complementary sequence, and by continuously changing the temperature, the fluorescence PCR instrument can accurately capture the sudden change in fluorescence intensity at different Tm values, thereby determining the corresponding nucleotide types.

[0022] 2. The combination of primers + molecular beacon probe used in the present invention belongs to the technical type of the second-generation PCR. Compared with the first-generation PCR technology commonly used in the current scheme, which still requires gel electrophoresis experiments to observe the target band, it has the natural generational difference advantage. Using the molecular beacon probe to hybridize and complement the amplified product, the obtained result has stronger specificity. By using the high-resolution signal collection system of fluorescence PCR to capture and analyze the fluorescence of the molecular beacon probe, its sensitivity is significantly improved compared with the traditional visual observation of the target band in gel electrophoresis.

[0023] 3. The method of the present invention realizes a closed-tube operation throughout the process, eliminates the nucleic acid aerosol pollution caused by opening the lid for sample loading in gel electrophoresis experiments, and ensures the accuracy of the results when conducting long-term and large-scale detections.

[0024] 4. The real-time fluorescence PCR instrument is utilized throughout the present invention, which can monitor the fluorescence changes during the amplification process and the melting curve process in real time, grasp the experimental dynamics in real time, and complete the determination of the genotype results of the FecB locus at the end of the experiment without the need for other subsequent experiments. It is simple, fast, and very suitable for application in large-scale detections.

[0025] 5. The detection method and detection kit of the present invention can be used to screen high-fertility genetic markers in breeding and production. By detecting the genotype of the FecB locus in individual sheep, it is determined whether they have a high-fertility genotype, and sheep with non-high-fertility genotypes in the screening population are optimized, ultimately achieving the purpose of improving economic benefits. Description of the Drawings

[0026] Figure 1 : Melting curve peak of sample A

[0027] Figure 2 : Melting curve peak of sample B

[0028] Figure 3 : Melting curve peak of sample C Detailed Embodiment

[0029] Example 1 Specific primers and molecular beacon probes of the present invention

[0030] According to the sheep BMPR1B gene in the NCBI database, by comparing sequences, it is determined that the FecB locus is located on the exon of g.431805 - 431997 in multiple transcripts, and the specific position is g.431965. The upstream and downstream sequences (SEQ ID NO.4) where the FecB locus is located are obtained according to the reference sequence NC_056059.1:

[0031]

[0032]

[0033] (The green-marked locus is the FecB locus)

[0034] By analyzing the SEQ ID NO.4 sequence, primers SEQ ID NO.1 and SEQ ID NO.2 were designed for template PCR amplification. According to the upstream and downstream sequences of the genomic region where the FceB site is located, a molecular beacon probe SEQ ID NO.3 covering this site was designed. The stem of the molecular beacon at both ends is designed as a hairpin structure formed by 6-base pairing. In the free state, the hairpin structure is intact, and the fluorescence signal of the fluorophore at the 5' end of the molecular beacon is captured by the quencher group at the 3' end, so the molecular beacon has no fluorescence and maintains a low fluorescence background. In the annealing state, the loop of the molecular beacon is complementary to the target gene fragment, opening the stem hairpin structure, resulting in the fluorophore being far from the quenching group, and the latter cannot completely capture the fluorescence. At this time, the molecular beacon emits fluorescence.

[0035] During the reaction process, first, a number of gene fragments covering the FecB site were generated by polymerase chain reaction. After the PCR amplification was completed, high-temperature denaturation was carried out to dissociate the PCR double strands and make them free in the reaction system. Subsequently, during the cooling process, the molecular beacon SEQ ID NO.3 was complementary to the target gene fragment and bound to emit fluorescence. Before the melting curve program started, its fluorescence value was the highest. Finally, as the temperature increased, the molecular beacon dissociated from the complementary target fragment and returned to the hairpin structure with complementary stems at both ends. The quencher group could effectively quench the fluorophore, and the fluorescence value decreased significantly, forming a melting peak.

[0036] Due to the A>G mutation at the FecB site, the same molecular beacon can form complete and incomplete complementarity with target fragments of different genotypes, resulting in melting peaks with different Tm values for different genotypes during the melting curve process: when the melting curve peak is at 53 - 56 °C, it is the C.746nt AA type, that is, the ++ site; when the melting curve peak is at 46 - 49 °C, it is the C.746nt GG type, that is, the BB site; when there are the above two melting curve peaks, it is the C.746nt AG type, that is, the B+ site.

[0037] Example 2: The nucleotide sequences of the specific primers and molecular beacon probes of the present invention are shown in Table 1 below:

[0038] Table 1

[0039]

[0040] Example 3: The detection method of the present invention

[0041] Step 1: Extract total DNA from the sample (sheep ear tissue or whole blood):

[0042] (1) Take an ear tissue sample the size of a soybean. After cutting it into small pieces as much as possible, use a glass mortar to grind it in a 1.5 ml EP tube, and then transfer it to a 2 ml EP tube; Wash the forceps, scissors, and mortar with 75% alcohol and dry them after processing each sample.

[0043] (2) Add 800 μl of lysis buffer and 30 μl of proteinase K (20 mg / ml) to the EP tube and mix well.

[0044] (3) Incubate the sample in an incubator at 55 °C overnight, and mix the EP tube from time to time until there are no tissue pieces in the tube.

[0045] (4) Add 800 μl of Tris-saturated phenol to the EP tube, invert and mix for 3 min, and centrifuge at 4 °C and 12000 rpm for 10 min.

[0046] (5) Take 650 μl of the supernatant, add 400 μl of Tris-saturated phenol and 400 μl of chloroform:isoamyl alcohol (24:1), invert and mix for 3 min, and centrifuge at 4 °C and 12000 rpm for 10 min.

[0047] (6) Take 550 μl of the supernatant, add 800 μl of chloroform, invert and mix for 3 min, and centrifuge at 4 °C and 12000 rpm for 10 min.

[0048] (7) Take 450 μl of the supernatant, add 800 μl of absolute ethanol, mix and shake for 6 min, and centrifuge at 4 °C and 12000 rpm for 10 min.

[0049] (8) Carefully discard the supernatant, leaving the DNA precipitate. Add 400 μl of 70% ethanol and pipette, centrifuge at 4 °C and 12000 rpm for 5 min, discard the supernatant, and repeat this step once.

[0050] (9) Place the EP tube in a fume hood and dry it until there are no small droplets in the tube.

[0051] (10) Dissolve the DNA by adding 50 - 150 μl of ultrapure water according to the size of the DNA mass, and store it at -20 °C.

[0052] Step 2: Preparation of reaction solution

[0053] Warm the components in the kit to room temperature for 20 minutes, and prepare a single reaction system according to the components described in Table 2 below.

[0054] Table 2:

[0055]

[0056] The specific reaction system mixture is prepared as shown in Table 3:

[0057] Table 3:

[0058]

[0059] Place the configured reaction system mixture in a PCR reaction tube.

[0060] Step 3: Sample addition

[0061] Open the lid of the PCR reaction tube, add 5.0 μL of the sample nucleic acid obtained in Step 2, and also add 5.0 μL for the blank control. Record the sample addition order.

[0062] Step 4: PCR amplification and melting curve analysis program

[0063] Set the relevant parameters for nucleic acid amplification of the instrument according to Table 4 below

[0064] Table 4:

[0065]

[0066] Note: The operations of different instruments vary. Please follow the instructions for melting curve analysis of fluorescence PCR for setting.

[0067] Step 5: Result interpretation

[0068] Through the analysis of the detection results of the PCR process and the collected fluorescence melting curve signals, the melting curve peak diagram and its corresponding gene polymorphism results are shown in Table 5.

[0069] Table 5:

[0070]

[0071] For the detection of the FecB locus, select SYBR / FAM for the fluorescence signal. When the melting curve peak is at 53 - 56 °C, it is the C.746nt AA type, that is, the ++ locus; when the melting curve peak is at 46 - 49 °C, it is the C.746nt GG type, that is, the BB locus; when there are the above two melting curve peaks, it is the C.746nt AG type, that is, the B+ locus.

[0072] Detection of three sheep samples in Example 4

[0073] Take ear tissue samples from three sheep and perform the experimental operations in the previous text: nucleic acid extraction to obtain three nucleic acid samples (labeled as sample A, sample B, and sample C respectively), reaction solution preparation, sample addition, and on - machine detection to obtain the following results: The melting curve peak of sample A is as Figure 1 shown; the melting curve peak of sample B is as Figure 2 shown; the melting curve peak of sample C is as Figure 3 shown;

[0074] According to the result determination criteria mentioned above (when the melting curve peak is at 53 - 56 °C, it is the C.746nt AA type, i.e., the ++ site; when the melting curve peak is at 46 - 49 °C, it is the C.746nt GG type, i.e., the BB site; when there are the above two melting curve peaks, it is the C.746nt AG type, i.e., the B+ site), the results of the samples are statistically analyzed as shown in Table 6.

[0075] Table 6:

[0076]

[0077] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A specific primer and molecular beacon probe composition for detecting the polymorphism of the FecB locus of the BMPR1B gene in sheep, characterized in that, The described composition includes an upstream primer with nucleotides as shown in sequence SEQ ID NO.1; a downstream primer with nucleotides as shown in sequence SEQ ID NO.2; a molecular beacon probe with nucleotides as shown in sequence SEQ ID NO.3; the 5'-end of the molecular beacon probe is labeled with a fluorophore and the 3'-end is labeled with a quencher; the FecB site polymorphism is generated by an A>G mutation at 746 nt in the coding region of the BMPR1B gene.

2. The specific primer and molecular beacon probe composition for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene according to claim 1, characterized in that, The 5'-end of the described molecular beacon probe is labeled with a FAM fluorophore and the 3'-end is labeled with a BHQ1 quencher.

3. A method for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene based on molecular beacon-melting curve, characterized in that, The described method includes the steps of: 1) Extracting the total DNA of the test sample; 2) Preparing a fluorescence quantitative PCR reaction system using the composition described in claim 1 or 2, adding the total DNA of the sample for detection; 3) Setting the reaction conditions; 4) Analyzing the melting curve after the reaction ends; 5) Judging the genotype of the FecB site of the sheep BMPR1B gene according to the melting curve peak.

4. The method for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene according to claim 3, wherein The reaction conditions are: pre-denaturation at 95°C for 180 s, 1 cycle; PCR at 95°C for 10 s, 60°C for 30 s, 72°C for 30 s, 40 cycles; denaturation at 95°C for 120 s, 1 cycle; annealing at 40°C for 120 s, 1 cycle; melting curve at 40°C - 65°C for 1 cycle.

5. The method for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene according to claim 4, characterized in that, When the melting curve peak is at 53 - 56°C, the genotype is judged as C.746nt AA type; when the melting curve peak is at 46 - 49°C, the genotype is judged as C.746nt GG type; when there are the above two melting curve peaks, the genotype is judged as C.746nt AG type.

6. The method for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene according to claim 3, wherein The described test sample is sheep ear tissue or sheep whole blood.

7. A kit for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene based on the molecular beacon-melting curve method, characterized in that, The described kit contains the specific primer and molecular beacon probe composition described in claim 1 or 2.

8. The kit for detecting the polymorphism of the FecB locus of the sheep BMPR1B gene according to claim 7, characterized in that, The kit further includes an enzyme mixture and a positive sample control; the enzyme mixture contains: Taq enzyme, dNTP, and Mg 2+ .

9. Use of the method for detecting the polymorphism of the FecB site of the sheep BMPR1B gene described in claim 3 in screening genetic markers for high fecundity in sheep.

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